Npn-1 Contributes to Axon-Axon Interactions That Differentially Control Sensory and Motor Innervation of the Limb

Abstract

The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs. During embryonic development, growing axons establish intricate neural networks with their peripheral targets, a process that builds the basis for complex behaviors. While wiring up the proper circuits in peripheral limbs, for example, motor axons from the spinal cord and sensory axons from the dorsal root ganglia converge in the spinal nerve. Here, they intermingle and are subsequently sorted before reaching the plexus region, the pivotal dorsal-ventral choice point on their path to the limb. In this study, we analyzed the contribution of the axon guidance receptor Neuropilin-1 (Npn-1) to determine how axons choose their path, how well they are able to maintain their correct path, and how it influences the interactions between spinal sensory axons and motor axons. We find that when Npn-1 is eliminated from sensory neurons, both sensory and motor axons are “derailed” from their correct nerve bundles, and there is a break in the tight coupling between these axonal populations. Loss of Npn-1 in motoneurons, however, leads to impairments in axon bundling and pathfinding errors only in motor axons, while sensory axons remain unaffected. Genetic ablation studies of either sensory or motor neurons corroborate the results on the mutual dependency and specificity of the outgrowing spinal projections. These results reveal a role for Npn-1 in controlling specific axon-axon interactions that lead to formation of proper spinal sensory-motor trajectories to the limb. Furthermore, they suggest that the presence of minimal numbers of sensory or motor axons is sufficient for the formation of correct spinal projections.